Modularity and diversity of target selectors in Tn7 transposons

Faure, Guilhem; Saito, Makoto; Benler, Sean; Peng, I-Feng; Wolf, Yuri I.; Strecker, Jonathan; Altae-Tran, Han; Neumann, Edwin N.; Li, David; Makarova, Kira S.; Macrae, Rhiannon K.; Koonin, Eugene V.; Zhang, Feng

doi:10.1016/j.molcel.2023.05.013

Cited by 15 publications

(6 citation statements)

References 69 publications

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“…Although there is currently no direct evidence to support this hypothesis, recent experiments with a native type V-K CAST system in cyanobacteria indicate that expression of Cas12k and TnsB are regulated by a CvkR transcriptional repressor ( 30 ), a feature that could be important in modulating the choice between targeted and untargeted transposition pathways. Considering the highly conserved operonic nature of TnsB, TnsC, and TniQ in Tn 7 -like elements, Tn 5053 , and type V-K CASTs, tight regulation in the stoichiometry of these proteins could be important for accessing an RNA-independent untargeted pathway ( 13 ). Future studies will be necessary to investigate this hypothesis further by deep sequencing bacteria with native type V-K elements to ensure that these rare events are not missed.…”

Section: Discussionmentioning

confidence: 99%

“…Although insertion specificity is often dictated by a single recombinase enzyme ( 2 , 3 ), some transposons encode heteromeric transposase complexes that distribute DNA target and integration activities across multiple distinct molecular components ( 4 , 5 ). Tn 7 -like transposons are unique in this regard, in that they have evolved to exploit diverse molecular pathways for target site selection, including site-specific DNA-binding proteins ( 6 ), replication fork–specific DNA-binding proteins ( 7 – 9 ), CRISPR RNA–guided DNA binding complexes ( 10 – 12 ), and additional DNA targeting pathways that have yet to be characterized ( 13 ). CRISPR-associated transposases (CASTs), in particular, represent both a fascinating example of CRISPR-Cas exaptation and an opportune starting point for the development of next-generation tools for programmable, large-scale DNA insertion ( 14 , 15 ).…”

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confidence: 99%

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Mechanism of target site selection by type V-K CRISPR-associated transposases

George,

Acree,

Park

et al. 2023

Science

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CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, “untargeted” transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo–electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Mechanism of target site selection by type V-K CRISPR-associated transposases

George,

Acree,

Park

et al. 2023

Science

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“…This includes the commonly used Scytonema hofmanni type V-K CAST, 39 a large variety of type I-F CAST systems, 34,40,41 as well as some type I-B and I-D CAST systems. [42][43][44] These and other systems can provide alternatives with different PAM preferences, transposition mechanisms, and insertion behaviors, expanding the possibilities for applying CAST systems as transcriptional perturbators.…”

Section: Discussionmentioning

confidence: 99%

Targeted Transcriptional Activation using a CRISPR-Associated Transposon System

Elizondo,

Chappell

2023

Preprint

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Synthetic perturbation of gene expression is central to our ability to reliably uncover genotype-phenotype relationships in microbes. Here, we present a novel gene transcription activation strategy that uses theVibrio choleraeCRISPR-Associated Transposon (CAST) system to selectively insert promoter elements upstream of genes of interest. Through this strategy, we show robust activation of both recombinant and endogenous genes across theE. colichromosome. We then demonstrate precise tuning of expression levels by exchanging the promoter elements being inserted. Finally, we demonstrate that CAST activation can be used to synthetically induce ampicillin-resistant phenotypes inE. coli.

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“…Additionally, the bioinformatic analysis of genomes has uncovered a diversity of CAST systems that could be adapted for our approach. This includes the commonly used Scytonema hofmanni-type V–K CAST, a large variety of type I–F CAST systems, ,, as well as some type I–B and I–D CAST systems. − These and other systems can provide alternatives with different PAM preferences, transposition mechanisms, and insertion behaviors, expanding the possibilities for applying CAST systems as transcriptional perturbators.…”

Section: Discussionmentioning

confidence: 99%

Targeted Transcriptional Activation Using a CRISPR-Associated Transposon System

Garza Elizondo,

Chappell

2023

ACS Synth. Biol.

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Synthetic perturbation of gene expression is central to our ability to reliably uncover genotype−phenotype relationships in microbes. Here, we present a novel transcription activation strategy that uses the Vibrio cholerae CRISPR-Associated Transposon (CAST) system to selectively insert promoter elements upstream of genes of interest. Through this strategy, we show robust activation of both recombinant and endogenous genes across the Escherichia coli chromosome. We then demonstrate the precise tuning of expression levels by exchanging the promoter elements being inserted. Finally, we demonstrate that CAST activation can be used to synthetically induce ampicillin-resistant phenotypes in E. coli.

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Modularity and diversity of target selectors in Tn7 transposons

Cited by 15 publications

References 69 publications

Mechanism of target site selection by type V-K CRISPR-associated transposases

Mechanism of target site selection by type V-K CRISPR-associated transposases

Targeted Transcriptional Activation using a CRISPR-Associated Transposon System

Targeted Transcriptional Activation Using a CRISPR-Associated Transposon System

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